Journal: Nature Communications
Article Title: A CDC42EP4/septin-based perisynaptic glial scaffold facilitates glutamate clearance
Figure Lengend Snippet: ( a ) Co-IP/IB assay of CDC42EP4 with representative septin subunits and GLAST from WT and KO cerebellar lysates. (Input) IB for SEPT4, SEPT7, SEPT2 and GLAST, respectively, detected a quadruplet of 54, 52, 48 and 44 kDa, a doublet of 51 and 48 kDa, a single 42 kDa band and a broad 55 kDa band in the cerebellar lysate. (IP) Anti-CDC42EP4 antibody pulled down SEPT4, SEPT7, SEPT2 and GLAST only from WT cerebellar lysate. The graphs show densitometric quantification of the yield ( n =3, *** P <0.001, ** P <0.01, * P <0.05, NS, P >0.05 by one-way ANOVA with post hoc Tukey test). (Note: the extraction condition including the lysis buffer composition was optimized to detect GLAST, which was distinct from the one used mainly for the proteomic analysis . See Methods.) ( b – d ) Pellet/supernatant assay results on the quantity and extractability of SEPT7, SEPT4 and GLAST in WT and KO cerebella. There was no significant difference in their amount and partitioning by genotype ( n =3, NS, P >0.05 by t -test). The same membranes were reprobed for α-tubulin as a loading control, which was used for normalization. ( e ) Double-label IF for GLAST (green) and CDC42EP4 (red) in WT cerebellar cortex showing their partial co-localization in Bergmann glial processes. Scale bars, 20 and 5 μm.
Article Snippet: We used antibodies for septins, SEPT2 (1:2,000), SEPT4 (1:3,000) and SEPT7 (1:4,000) as previously described , GLAST , 3-phosphoglycerate dehydrogenase (Phgdh) , calbindin , carbonic anhydrase 8 (Car8) , VGluT1, 2 (ref. ) and commercial antibodies for GluR1, 2 (Alomone Labs, AGC-004, 1:200, AGC-005, 1:150), GLAST (Frontier Institute, Rb-Af660, 1:2,000), PSD-95 (Cell Signaling, 3450, 1:1,000), CDC42 (Santa Cruz, L0809, 1:100), β-actin (Sigma, A5441, 1:5,000) and α-Tubulin (Sigma, T9026, 1:10,000).
Techniques: Co-Immunoprecipitation Assay, Lysis